I. Pharmacology of the human BRS-3 receptor. Three different receptors including the gastrin-releasing peptide receptor (GRP-R), the orphan receptor, BRS-3 and neuromedin B receptor (NMB-R), mediate the action of bombesin-related peptides in mammals. At present no ligands exist for the BRS-3 receptor, its natural ligand is unknown as is its cellular basis of action and pharmacology. In order to begin to determine each of these, we have used two different strategies to prepare cell lines expressing hBRS-3 receptors that should reflect their true pharmacology and coupling. We discovered a synthetic Bn analogue [DPhe-6,$-Ala-11,Phe-13,Nle-14] Bn(6-14) that has high affinity for the BRS-3 receptor. An analogue [DTyr-6,$Ala-11,Phe-13,N6-14]Bn(6-14) was developed which could be used for ligand binding studies. The BRS3R was found to be a G-protein coupled receptor, with a unique pharmacology demonstrating it interacted with no known natural bombesin peptides with high affinity suggesting its natural ligand was either a new undiscovered peptide or a novel Bn peptide. Binding studies identified two classes of antagonists that may be useful for investigating the role of this receptor in physiologic processes. With these cell lines and the discovery of a high affinity ligand which functions as an agonist, detailed studies of the hBRS-3 cell biology can now be performed. II.b. Molecular basis of action of gastrin-releasing peptide receptor (GRP-R) antagonists. Most G coupled-receptors have only high affinity nonpeptide antagonists. The GRP-R is unique in having 6 classes of high affinity peptide antagonists. Whether peptide antagonist's receptor sites of interaction differ from agonists is unknown. To determine this we used site-directed mutagenesis and made GRP-R chimeras using the closely-related NMB-R. The specificity of the high affinity GRP-R antagonist, [DPhe-6]Bn(6-13) methyl ester (ME) for the GRP-R (1000X) was due almost entirely to the 4th extracellular loop (EC) of the GRP-R whereas the selectivity of agonists was not. A detailed study involving single and combination point mutations of the 4th EC loop identified the 4 different amino acids in the 4th EC loop determining this high affinity. A model is being developed based on these amino acids of the binding packet for this antagonist. II.c. Effect of GRP-R receptor number on cellular processes. GRP-R's can vary 1000-fold in number on different natural occurring cells and the significance of this is unknown and has been poorly studied, as with most PLC-coupled receptors. To address this issue we made three different cell lines with GRP-R varying by 300-fold over the range seen in natural cells. GRP-R number had no effect on binding affinity, potency for PLC activation or tyrosine phosphorylation of p125FAK or receptor internalization rate/extent. In contrast it altered ligand degradation rate, extent of down-regulation, desensitization, and efficacy for PLC activation. These results demonstrate that the effect of receptor number differs for PLC-coupled receptors from that reported for adenylate cyclase coupled receptors and differs for different GRP-R mediated cellular functions. This may have important implications especially for growth-related functions, which can be regulated by the extent of receptor down-regulation or desensitization.